Pharmaceutical compositions comprising Aloe pectins, and methods for their production and use

ABSTRACT

Pectins having a combination of unexpectedly high molecular weights and low degrees of methylation have been isolated from Aloe vera plants, are superior gel pectins for forming calcium cross-lined gels. Such pectins can be obtained by extracting homogenized Aloe Vera plants or portions thereof. These pectins can be used to prepare pharmaceutical compositions comprising pharmacological agents for application to animals and humans, so as to provide controlled release of the pharmacological agent.

RELATED APPLICATION DATA

This application claims the priority of each of the U.S. patentapplications, recited in this paragraph, and is a divisional applicationof U.S. Utility application Ser. No. 10/422,867 filed Apr. 23, 2003, nowallowed, which was a continuation of U.S. Utility application Ser. No.09/352,610, filed Jun. 2, 1999 and now granted as U.S. Pat. No.7,022,683, which was a divisional application of U.S. Utilityapplication Ser. No. 09/078,204, filed May 13, 1998, now granted as U.S.Pat. No. 5,929,051. The disclosure of all the above-recited parentapplications is hereby incorporated herein in their entirety by thisreference.

INTRODUCTION

This invention relates to pectins. More specifically, this inventionrelates to Aloe pectins, process of isolation and their use.

Following abbreviations are used:

Ab, antibody; AG, arabinogalactan; APase, alkaline phosphatase; CDTA,trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid; Da, dalton;DAc, degree of acetylation; DM, degree of methylation; EDTA,ethylenediaminetetraacetic acid; Gal, galactose; Gal A, galacturonicacid; GalNAc, N-acetylated galactosamine; Glc, glucose; Glc A,glucuronic acid; HM, high methoxyl; HMW, high molecular weight; HPSEC,high performance size exclusion chromatography; HR, hairy region; HT,heating; kDa, kiloDalton; LM, low methoxyl; LMW, low molecular weight;Man, mannose; MWCO, molecular weight cut-off; PBS, phosphate bufferedsaline (10 mM sodium phosphate, 150 mM NaCl, pH 7.4); RG,rhamnogalacturonan; RT, room temperature; SEC, size exclusionchromatography;SF, supercritical fluid; SR, smooth region;TMS,trimethylsilyl; TN buffer, 25 mM Tris, 150 mM NaCl, pH 7.4.

Pectin is a plant cell wall component. The cell wall is divided intothree layers, middle lamella, primary, and secondary cell wall. Themiddle lamella is the richest in pectin. Pectins are produced anddeposited during cell wall growth. Pectins are particularly abundant insoft plant tissues under conditions of fast growth and high moisturecontent. In cell walls, pectins are present in the form of a calciumcomplex. The involvement of calcium cross-linking is substantiated bythe fact that chelating agents facilitate the release of pectin fromcell walls.

Pectin is a complex polysaccharide associated with plant cell walls. Itconsists of an α1-4 linked polygalacturonic acid backbone intervened byrhamnose residues and modified with neutral sugar side chains andnon-sugar components such as acetyl, methyl, and ferulic acid groups.Based on the current understanding, the general structure of pectins orpectic substances is shown in FIG. 1. The overall structure is shown ontop, while the detailed structure is shown on the bottom. The neutralsugar side chains which include arabinan and arabinogalactans (Types Iand II) are attached to the rhamnose residues in the backbone at the O-3or O-4 position. The rhanmose residues tend to cluster together on thebackbone. So with the side chains attached this region is referred asthe hairy region and the rest of the backbone is hence named the smoothregion. Rhanmose residues are 1-2 linked to Gal A residues in thebackbone and the configuration of this linkage has now been determinedto be α.

Pectins are traditionally used as food additives. However, their use hasextended into pharmaceutical areas as well. Pectins have long been usedas an anti-diarrhea agent and can improve intestinal functions. Theanti-diarrhea effect is thought to be in part due to pectin'santi-microbial activity.

Pectins are also effective against gastrointestinal ulcers andenterocolitis. Pectins also influence cell proliferation in theintestines. They also have a blood cholesterol-lowering effect andexhibit inhibition of atherosclerosis. This effect is the result ofinteractions between pectins and bile salts. Pectins have also beenshown to affect the fibrin network in hypercholesterolaemic individuals.

The ability to interact with many divalent metal ions renders pectins astrong detoxifying agent. It has been shown that pectins are effectivein removing lead and mercury from the digestive tract and respiratoryorgans. Lately, pectins have been found to be effective for thetreatment of heartburn caused by esophagus acid reflux.

Recently, so-called modified citrus pectins, which are small molecules(˜10 kDa) obtained by alkaline degradation, have been found to beeffective in the prevention of cancer cell metastasis in laboratoryanimals.

Because of the presence of neutral sugar side chains and some othernon-sugar components, the structure of pectins is very complex;essentially no two molecules have identical structures, which is thereason why pectin is often described using the term “pectic substances”.Pectic substances is commonly used to encompass pectin, pectic acid andits salts (pectates), and certain neutral polysaccharides (arabinan,arabinogalactan, and galactan). Pectic acids or pectates aredeesterified pectins.

Rhanmose, galactose, arabinose, and xylose are the most common neutralsugar components of pectins. The less common ones are glucose, mannose,and fucose. Some of the xylose residues are individually attached to GalA residues at O-3 position. Three types of neutral sugar side chainshave been identified in pectins. Arabinan consists of α1-5 linkedarabinose. Arabinogalactan I consists of β1-4 linked galactose withshort arabinan chains attached at O-3. In arabinogalactan II, galactoseis β1-3&6 linked with arabinose attached.

Methylation occurs at carboxyl groups of Gal A residues. The degree ofmethyl-esterification is defined as the percentage of carboxyl groups(Gal A residues) esterified with methanol. A pectin with a degree ofmethylation (“DM”) above 50% is considered a high methoxyl (“HM”) pectinand one with a DM<50% is referred to as low methoxyl (“LM”) pectin. Mostof the natural pectins are HM with a few exceptions such as sunflowerpectin. The degree of acetylation (DAc) is defined as the percentage ofGal A residues esterified with one acetyl group. It is assumed that onlythe hydroxyl groups are acetylated. Since each Gal A residue has morethan one hydroxyl group, the DAc can be above 100%. DAc is generally lowin native pectins except for some such as sugar beet pectin.

Pectin may contain some non-sugar components. Ferulic acid esters havebeen found in sugar beet pectin. They are linked to the arabinose andgalactose residues in the neutral sugar side chains.

Pectins are soluble in water and insoluble in most organic solvents.Pectins with a very low level of methyl-esterification and pectic acidsare only soluble as the potassium or sodium salts. As for otherpolymers, there is no saturation limit for pectins, but it is difficultto obtain a true solution with concentrations higher than 3-4%.Commercial pectins have a size range of 7-14×10⁴ Da. Citrus pectins arelarger than apple pectins. Viscosities of pectin solutions are generallylow and so pectins are seldom used as thickening agents. The viscosityis directly related to the size, pH, and also to the presence ofcounterions. Addition of monovalent cations reduces viscosity.

Pectins can interact with several divalent metal ions. The order ofselectivity is Cu˜Pb>>Zn>Cd˜Ni≧Ca. This activity is the basis forpectin's detoxification effect.

The Gal A residues in the pectin backbone are α1-4 linked. Both hydroxylgroups of D-Gal A at carbon atoms 1 and 4 are in the axial position. Theresulting linkage is therefore trans 1-4. This type of linkage resultsin increased chain stiffness of the polymer. So pectin with aflexibility parameter B between 0.072-0.017 are rigid molecules. It hasbeen suggested that the insertion of rhamnose residues in the backbonecause a T-shaped kink in the backbone chain. An increase in rhamnosecontent leads to more flexible molecules. Pectins can be considered as azigzag polymer with long and rigid smooth regions and flexible hairyregions (rich in rhamnose) serving as rotating joints. The DM also hascertain effects on chain flexibility. In solution, pectin molecules havebeen shown to assume a right-handed helical structure.

Pectins are most stable at pH 3-4. Below pH 3, methoxyl and acetylgroups and neutral sugar side chains are removed. At elevatedtemperatures, these reactions are accelerated and cleavage of glycosidicbonds in the galacturonan backbone occurs. Under neutral and alkalineconditions, methyl ester groups are saponified and the polygalacturonanbackbone breaks through β-elimination-cleavage of glycosidic bonds atthe non-reducing ends of methoxylated galacturonic acid residues. Thesereactions also proceed faster with increasing temperature. Pectic acidsand LM pectins are resistant to neutral and alkaline conditions sincethere are no or only limited numbers of methyl ester groups.

There are many enzymes that can specifically modify and degrade pectinmolecules. These enzymes include endo- and exo-polygalacturonase(EC3.2.1.15 and EC 3.2.1.67), pectate lyase (EC 4.2.2.10), pectinmethylesterase (EC 3.1.1.11), pectin acetylesterase, andrhamnogalacturonase. Endo-polygalacturonase is specific fornon-esterified α1-4 linked Gal A residues and requires four adjacentnon-esterified Gal A residues to function. This enzyme can be producedby plants, fungi, and bacteria.

Both HM and LM pectins can form gels, but by totally differentmechanisms. HM pectins form gels in the presence of high concentrationsof co-solutes (sucrose) at low pH. LM pectins form gels in the presenceof calcium. In addition, the sugar beet pectin can form gels throughcross-linking of the ferulated groups.

The calcium-LM pectin gel network is built by formation of the “egg-box”junction zones in which Ca++ ions cause the cross-linking of twostretches ofpolygalacturonic acids. In apple and citrus pectins,stretches of polygalacturonic acids without rhamnose insertion have beenestimated to be as long as 72-100 residues. The zone is terminated bythe rhamnose residue in the backbone. The calcium-LM pectin gel isthermoreversible. The calcium can therefore be added at the boilingpoint and gel formation occurs upon cooling. It is possible to obtain afirm resilient gel with 0.5% pectin and 30-60 mg/g Ca++. A high contentof pectin with little calcium gives an elastic gel whereas a highcalcium concentration with a minimum of pectin results in a brittle gel.

Addition of monovalent counterions enhances the calcium-LM pectin gelformation, i.e., less calcium is required for gel formation.

Commercial pectins are mainly extracted from apple pomace or orangepeels under hot acid conditions followed by alcohol precipitation. Theraw materials are first blanched, then washed to inactivate endogenousenzymes capable of degrading pectins, and to remove pigments. A commonmethod for enzyme inactivation is alcohol treatment, i.e., cell wallfibers are prepared as the so-called alcohol insoluble residues (“AIR”)or solids (“AIS”).

Various extraction conditions have been used for isolation of pectinsfrom plant cell walls. These include use of chelating agents such asEDTA, CDTA, sodium hexametaphosphate and ammonium oxalate at pH 3-6.5,hot dilute acid (HCl, pH 1.5-3), and cold dilute base (NaOH and Na₂CO₃;pH 10). The extraction is often performed at elevated temperatures(60-100° C.) to increase the yield. Commercial citrus and apple pectinsare extracted with hot dilute acid. Since pectins are readily degradedat a pH of <3, the extraction process usually lasts briefly depending onthe temperature used. The pH of 3-6.5 at which the chelating agents areused is below the pH needed for their optimal chelating effect, but isused to minimize the pectin degradation through β-elimination. Like hotdilute acid extraction, the alkaline extraction can cause extensivedegradation. It is only performed at 0-4° C. in order to minimize thedegradation through β-elimination. The cold alkaline extraction is oftenused as the last step of a sequential extraction to remove those pectinstightly bound to cell walls.

Enzymes have also been examined for pectin extraction. They includearabinase, galactanase, polygalacturonase, and rhamnogalacturonase. Thepolygalacturonase-producing yeast cells have also been used directly forpectin extraction.

Characteristics of pectins extracted under different conditions mayvary. Pectins extracted at elevated temperatures are smaller than thoseobtained at room temperature and richer in neutral sugars. The smallersize is the result of degradation under the harsher conditions. However,the yield is much higher at elevated temperature. Those pectins obtainedwith a chelating agent usually have a higher Gal A content. The pectinsobtained under the cold alkaline conditions generally have a reduced GalA content and a higher neutral sugar content.

Industrial pectins, either HM or LM, are mainly obtained from apple andcitrus by acid extraction and alcohol precipitation. LM pectins areobtained from HM ones by chemical de-esterification. Pectins have afavorable regulatory status as a food additive. They are classified asGenerally Recognized As Safe (“GRAS”) in the United States andAcceptable Daily Intake (“ADI”) in Europe. That is, its use is onlylimited by current Good Manufacturing Practice (“cGMP”) requirements tomeet certain specifications. These specifications include a minimal GalA content of 65% (w/w).

HM pectin can be converted into a different type of LM pectin, i.e.,amidated pectin. This is achieved by treating HM pectin with ammoniaunder alkaline condition in alcoholic suspensions. The methyl estergroups are replaced with amide groups. The amidated pectin has a bettergel formation ability in the presence of calcium as compared to theregular LM pectin.

Many other plant sources have also been examined for pectin production.Two of them, sugar beet pulp and sunflower head, have been studiedextensively. Both are abundant as raw materials. However, sugar beetpectin has a poor gel forming ability largely due to its high acetylgroup content and small molecular size (˜5×10⁴ Da). The sunflowerpectins are naturally LM and can be efficiently extracted with chelatingagents. They often suffer from poor quality of raw materials and poorcolor quality (usually tan) of the pectin end products.

Pectins from different plant sources have different characteristics. Ingeneral, all commercial pectins including those that have gone throughfurther processing have a certain degree of coloration as a finalproduct. The color ranges from light yellow/brown (citrus pectin) todark tan (apple and sunflower head pectins). The coloration is caused bythe combination of two factors: natural color (pigmentation) of the rawmaterials and their content of polyphenols. Chemically, sunflower headpectin has a very high Gal A content and is a natural LM pectin, whereassugar beet pectin has a relatively low Gal A content and a very highcontent of acetyl and ferulic acid groups. The structures of apple andcitrus pectins are very similar to each other.

A set of techniques has been established for pectin analysis. The Gal Acontent is determined by the method using m-hydroxyldiphenyl for colorformation. This assay is simpler than previous assays and has minimalinterference from neutral sugars. Other assays for Gal A determinationhave also been described. Sugar compositions are analyzed by GLC orGC-MS using alditol acetate or trimethylsilylether (“TMS”)derivatization. GLC procedures are most often used to determine methylester content, which involves saponification with base (0.5N) andmeasurement of methanol by GLC on a Poropak Q columan at 120° C. or aCarbowax 1500 column at 125° C. A capillary electrophoresis method hasalso been examined for determining DE of pectins. A rapid and sensitivecalorimetric assay is used to measure the acetyl groups.

The size determination is achieved by various means which includeviscosity, HPSEC, and gel permeation chromatography. Recently, lightscattering has been proposed as a more accurate method. The intrinsicviscosities of pectins are often determined using the Ubbelohdeviscometer. This is done in the presence of 0.1-0.15 M NaCl due to theelectrolytic nature of pectin molecules.

The purification of pectins is mostly achieved by ion exchangechromatography and cupric precipitation. For ion exchangechromatography, DEAE sepharose CL-6B matrix and acetate buffer (pH 4.8)are most widely used. The neutral sugar content of pectins is determinedfollowing purification with these methods.

SUMMARY

Broadly, one aspect of the present invention pertains to an Aloe pectinhaving at least one of the following properties: degree of methylationof less than about 50% by mole; rhamnose content of from about 2 toabout 15% by mole; 3-0-methyl rhamnose content of from about 0.1 toabout 5% by mole; and capable of forming a gel in the presence of asolution of a calcium salt; the Aloe pectin are isolated from the leafof an Aloe by extraction, wherein the extraction is accomplished by asupercritical fluid, a water-soluble organic solvent, an acid, analkali, a chelating agent, a bacteria, an enzyme, or a combinationthereof.

According to the present invention, pectins from gel and rind cell wallfibers of Aloe vera are extracted, isolated and identified. Serialtreatment of Aloe fibers with a chelating agent such as EDTA at a pH offrom about 7 to about 8.5 is most efficient method of extraction.Purified Aloe pectins are obtained by further treating Aloe pectin withan ion exchange resin. Aloe pectins contain galacturonic acid, anunusually high level of rhamnose, and 3-OMe-rhamnose. Two classes ofAloe pectin distinguished by size are obtained: the room temperatureextraction generated a high-molecular-weight (HMW) pectin whereasextraction with heating produced a low-molecular-weight (LMW) pectin.Aloe pectins naturally have a low methoxyl (LM) content. Both the HMWand LMW pectins are capable of gel formation in the presence of calcium.In addition, Aloe pectins, especially the HMW pectin, forms monovalentcation-based gels at low temperatures which revert back to solution whenbrought to room temperature. The HMW Aloe pectin-calcium gel is a highlyefficient encapsulating agent suitable for controlled release ofpharmacological substances, such as proteins, antibodies, and vaccines.Aloe pectins form a matrix for antigen and antibody precipitationreactions. Further Aloe pectins form a storage matrix forpharmacological substances. Aloe pectins from pulp exhibit an off-whitepowder color and produced clear solutions when dissolved in water.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a general structure of pectins or pectic substances, inwhich “HR” stands for hairy region, “SR” stands for smooth region, “AG”stands for arabinogalactan, and “RG” stands for rhamnogalacturonan.

FIG. 2 is a cross-section schematic representation of Aloe Vera leafstructure.

FIG. 3 shows the structural components of Aloe Vera pulp mesophyl cells.

FIG. 4 is a flow chart for sequential Aloe pectin extraction (at roomand high temperatures) using EDTA as the chelating agent.

FIG. 5 shows the use of Aloe pectin as an encapsulating agent forcontrolled release. The relative amounts of the enzyme released frompectin beads were measured with substrate PNPP:

(a) Spontaneous release in water in relation to pectin concentrationsshown with APase-Ab conjugate beads;

(b) Spontaneous release in water in relation to the sizes of Aloepectins shown with APase-Ab conjugate beads made with 10 mg/ml Aloepectins; and

(c) Effect of pH and NaCl (150 mM) in triggering release shown withAPase beads made with 15 mg/ml Aloe pectin(1.36×10⁶ Da). TN, 25 mM Trisand 150 mM NaCl, pH 7.4; saline, 150 mM NaCl.

DETAILED DESCRIPTION

Aloe vera has long been used as a medicinal plant. It is a succulentplant adapted to live in desert and semi-desert conditions. Thesucculents are characterized by their possession of abundant waterstorage tissues. The Aloe vera leaves consist of two parts, green rindand clear pulp (i.e., inner gel or inner fillet). The latter is thewater storage tissue and is most widely used for medicinal purposes.Because of its clear and slimy appearance, the pulp is often referred asthe mucilaginous gel which has largely been treated as a singlehomogenous entity.

Pectins or pectic substances from Aloe vera and their extraction havenot previously been described in any detailed manner. A pectic substancerich in Gal A has been described as the major pulp polysaccharidecomponent. This polysaccharide with a Gal A content of 85% was isolatedfollowing hot water extraction of the alcohol precipitates of clearpulp. Neutral sugar composition analysis detected galactose, rhamnose,arabinose and trace amounts of mannose, glucose, and xylose. Thisfinding was interpreted as a result of plant variation within the Aloevera species and specific local conditions, as compared to the fact thatmost other studies identified mannose-rich polysaccharides as the majorpolysaccharide component in the Aloe vera pulp. A Gal A-richpolysaccharide has been obtained through hot water and ammonium oxalateextractions from whole leaf materials previously treated with boilingmethanol. The Gal A content was estimated to be 55% based on paper andgas-liquid chromatography. This polysaccharide was degraded bypectinaseand hence identified as pectin which in turn was claimed to be the majorpolysaccharide of Aloe vera. In all the above studies, no linkagestudies on neutral sugars were performed, nor any detailedcharacterization of other chemical and physical properties (e.g., size,DM, DAc, and gel formation) of the isolated polysaccharides.

One aspect of this invention started from the clear pulp or filletedinner gel of the Aloe vera leaf. The isolation of different parts ofAloe leaf has been described in U.S. Pat. Nos. 4,735,935, 4,851,224,4,917,890, 4,959,214, and 4,966,892, the entire content of each of thesepatents is hereby incorporated by reference. The clear gel containslarge mesophyll (water storage) cells with very limited numbers ofdegenerative cellular organelles and the green rind contains muchsmaller cells which are rich in cellular organelles such as mitochondriaand chloroplasts. It was found that following homogenization, the pulpcould be separated into two major portions, soluble and insoluble. Thesoluble portion was shown to be rich in the α1-4 linked mannose. Theinsoluble portion mainly consisted of clear cell walls or cell wallfibers (based on its microscopic appearance under low magnificationfollowing homogenization). The cell wall component in Aloe vera pulpextracts has not been previously described. The cell wall fiberscontained a high level of Gal A (34% w/w), whereas the soluble portioncontained <5% (w/w) of Gal A. This data clearly suggested that thesecell wall fibers were potentially rich in pectin. Ensuing experimentsshowed that a large amount of pectin (as high as 50%, w/w) with anaverage Gal A content >70% (w/w) could be extracted from these pulp cellwall fibers. A large amount of pectin that is equally rich in Gal Acould also be extracted from the cell wall fibers isolated from therind. These pectins from pulp or rind fibers were named Aloe pectins.

The cell wall fibers were isolated by centrifugation or filtrationfollowing homogenization of the pulp or rind and used directly forpectin extraction without any treatment except for washing in water. TheAloe pectin could be extracted from these fibers using previouslydescribed methods, i. e., hot acid at a pH of ˜1.5, cold alkali (NaOH orNa₂CO₃) at a pH of ˜10, and chelating agents (EDTA, sodiumhexametaphosphate) at a pH of 4.0-6.5. However, the most efficientextraction method that gave the highest yield was found to be the use ofa chelating agent at a pH above 7 (7-8.5). The chelating agent used wasEDTA. The uniqueness of this extraction procedure was the higher pH(7-8.5) used, since in all previous studies, the chelating agent hasalways been used at a pH≦6.5 in order to minimize the degradationthrough β-elimination. The reason behind using this higher pH is thatAloe pectins are naturally LM (see below), a form of pectin resistant toβ-elimination under alkaline conditions, and EDTA functions mostefficiently at a pH above 7.

A two-step sequential extraction procedure maximized the use of fibersand yielded two types of pectin distinguished by size, HMW and LMW. Thefibers were extracted first at RT followed by another extraction underHT (up to 80° C.). The RT extraction produced the HMW pectin with anaverage MW of 1.1×10⁶ Da and the HT extraction produced the LMW pectinwith an average MW of 1.9×10⁵ Da. The MW was directly correlated to theintrinsic viscosities; the HMW pectin exhibited an intrinsicviscosity≧550 ml/g and as high as 978 ml/g. The MW and intrinsicviscosity of HMW Aloe pectin was much higher than those of thecommercial pectins.

Aloe pectin exhibited some distinct features in sugar compositions. Theycontained a high level of rhamnose; the rhamnose content in Aloe pectinswas at least 2 times higher than in other pectins, mainly citrus, apple,sugar beet, and sunflower. The rhamnose is a key sugar in the pectinbackbone whose content affects the flexibility of the molecule. Aloepectins also possessed a rare sugar, 3-OMe-rhamnose, which has not beendescribed in any other pectins. Aloe pectins were found to be naturallyLM, having a DM generally <30% and often <10%. They were capable of gelformation in the presence of calcium. Uniquely, Aloe pectins, especiallythe HMW ones, could form a monovalent cation (NaCl)-based reversible gelat low temperature (4° C.) at a very low pectin concentration (1 mg/ml).Such cold gelation has not been described for any other pectins.

The green rinds from Aloe vera leaves are generally removed as wasteduring production of pulp-based products. These rinds with small amountsof pulp remaining attached to them account for ˜60% (w/w, wet) of thewhole leaf. It was found that cell wall fibers prepared from these rindsproduced an Aloe pectin yield similar to those from pulp. The Aloepectins from rind were equally rich in Gal A and shared the sameproperties with the those from pulp, i.e., being naturally LM, high inMW and intrinsic viscosity (for HMW ones), and capable of calcium gelformation as well as the monovalent cation-based gel formation at lowtemperature (4° C.).

The Aloe pectins from the pulp fibers are off white powders as the endproducts and produced clear solutions as compared to the yellow to tanpowders and cloudy solutions of current commercial and experimentalpectins from citrus, apple, sugar beet, and sunflower. Those from therind fibers were light green-brownish powders and produced solutionsthat were cloudy, but to a lesser extent than the best citrus pectins.The powder color and solution clarity of Aloe pectins from rind fiberscould be substantially improved by additional alcohol rinsing.

Together, Aloe pectins are unique pectins and could be distinguishedfrom other pectins, i.e., citrus, apple, sugar beet, and sunflower, byone or more of the following characteristics:

-   -   1) A high molecular weight (>1×10⁶ Da) and a high intrinsic        viscosity (>550 ml/g).    -   2) A high rhamnose content.    -   3) Possessing 3-OMe-rhamnose.    -   4) Being naturally LM.    -   5) Capable of calcium gel formation.    -   6) Capable of monovalent cation-based gel formation at low        temperature (4° C.).    -   7) Off white powders and clear solutions (Aloe pectin from        pulp).

Materials and Methods

Materials

Aloe vera (Aloe Barberdensis Miller) plants (10″) were obtained from H&PSales, Inc. (Vista, Calif.) through Lowe's store. Bulk acetylated mannan(BAM) is an Aloe vera pulp extract of Carrington Laboratories, Inc.Various commercial pectins and polygalacturonic acid were used. Theyinclude HM citrus (P-9561 with a DM of 92% and P-9436 with a DM of 64%),LM citrus (P-9311 with a DM of 28%), polygalacturonic acid (P-1879) fromSigma Chemical Co., HM citrus (PE100 with a DM of 67%) from SpectrumChemical Co., and HM citrus (CU401) and apple (AU201) fromHerbstreith-Fox KG. Following reagents were also obtained from SigmaChemical Co.; disodium EDTA, tetrasodium EDTA, endo-polygalacturonase,alkaline phosphatase, alkaline phosphatase-antibody (IgG) conjugate,Folin-Ciocalteu's reagent, imidazole, and all neutral and acidic sugarsused. The alkaline phosphatase substrate pNPP was obtained from Pierce.Sodium hexametaphosphate was obtained from Fluka Chemie AG.

Generally, BAM may be prepared from Aloe leaves as follows:

1. Aloe leaves are washed, sliced open and filleted to remove the leafrind. The clean (substantially anthraquinones free) inner gel isretained while the green rind is discarded.

2. The filleted material is homogenized (creparo) and extensivelyfiltered with a Finisher Model 75 (FMC, Chicago, Ill.) to remove most ofthe pulp.

3. The clear viscous gel is acidified to a pH of approximately 3.2 withdilute HCl.

4. The acidified gel is then extracted with four volumes of 95% ethanolat ambient temperature. Floating material is removed, then thealcohol/water mixture is siphoned off while the solid precipitate iscollected by centrifugation. Most alcohol/water soluble substances suchas organic acids, oligosaccharides, monosaccharides, anthraquinones andinorganic salts are eliminated by the alcohol extraction process.

5. The solid Aloe vera extract is then washed with fresh alcohol,centrifuged, freeze dried, and ground to a white powder.

The product is stable at room temperature in the freeze-dried form forseveral years if protected from additional moisture. The detailedprocedures for producing substantially anthraquinone-free Aloe gel, forproducing substantially anthraquinone-free Aloe juice, for extractingactive chemical substance(s) from an Aloe leaf, for preparing BAM andfor extracting from an Aloe leaf substantially non-degradablelyophilized ordered linear polymers of mannose have been described inCarrington's U.S. Pat. Nos. 4,735,935, 4,851,224, 4,917,890, 4,957,907,4,959,214, and 4,966,892, the entire content of each of which isincorporated by reference. The uses of Aloe products have been describedin Carrington's U.S. Pat. Nos. 5,106,616, 5,118,673,5,308,838,5,409,703,5,441,943, and 5,443,830, the entire content of each of whichis hereby incorporated by reference.

EXAMPLE 1 Light and Electron Microscopy of Leaf Sections

Fresh Aloe vera leaves were sectioned with a surgical blade into 2-3mm-thick pieces. The sections were directly observed under the lightmicroscope (Olympus BH-2). For histological analysis, fresh Aloe veraleaves were fixed in 10% formalin in PBS and sections were stained withtoluidine blue.

The protocols for tissue fixing and staining for electron microscopyfollowed that described by Trachtenberg (Annuals of Botany, 1984, 53,pp. 227-236). Briefly, fresh pulp tissue blocks were fixed at roomtemperature in 4% glutaraldehyde in 0.2 M cacodylate-HCl buffer (pH 7.2)for 2 hrs followed by fixing for 2 hrs in 2% osmium tetroxide in thesame buffer. The tissues were dehydrated and sectioned after embeddingin resin. The tissue sections were stained with uranyl acetate, andexamined using a Zeiss 10C electron microscope. The light microscopy ofleaf sections showed that the pulp (3) consisted of large clearmesophyll cells, which exhibited a hexagonal shape (FIG. 2). The sizesof these cells were very large, often more than 300 μm in width. Thewalls of these cells were clear and transparent. The cells in the rind(1) were much smaller as compared to those in the pulp (3) (FIG. 2).Electron microscope examinations revealed, in addition to cell walls(6), liquid gel (7), only the cell membranes in the pulp along with verylimited number of degenerative cellular organelles (8) (FIG. 3). Nuclei,chloroplasts and other cellular organelles such as mitochondria wereonly observed in the green rind and vascular bundles, (2) (FIG. 2).

EXAMPLE 2 Light Microscopy of Cell Wall Fibers

BAM was dissolved in water at 2 mg/ml. The solutions were stirred atroom temperature for 3 hrs or at 4° C. for overnight. They were thencentrifuged at low speed (1000 rpm or 180 g) for 15 min (Beckman TJ-6).The pellet was collected, washed once with water, and dried (Centrivap,Labconco). The weight of pellet was determined following drying. A smallsample of the pellet was examined under the light microscope (OlympusBH-2). The insoluble pellet materials from the pulp extracts appeared tobe fibers at low magnification (4×), and to be clear transparent sheetsat higher magnification (10× and 40×) with an appearance identical tothose clear pulp cell walls described above. With less extensivehomogenization, some of these fibers still retained the originalstructural features of the mesophyll cells. These observations togetherindicate that the insoluble fibers are derived from the pulp mesophyllcell walls.

EXAMPLE 3 Extraction of Aloe Pectins

Preparation of cell wall fibers. Two types of cell wall fibers wereused, alcohol-treated and non-alcohol-treated. The alcohol-treatedfibers were isolated from BAM by centrifugation. BAM was dissolved inwater at 2 mg/ml. The solution was then centrifuged at 180 g for 10 min.The pellet, consisting of cell wall fibers, was harvested and washedthree times with water before being dried. Since BAM has gone throughalcohol precipitation, these fibers are therefore similar to thosealcohol insoluble residues or solids (AIS) that are commonly preparedfor extraction of pectins from other plant tissues.

The non-alcohol-treated fibers include the crude pulp and rind fibers.Crude pulp fibers were those retained by the coarse filtration duringproduction of BAM and other pulp-based products. They are the same asthose found in BAM, except for being larger in size and hotalcohol-treated. They were collected with a no. 18 sieve (1 mm opening)with minimal loss and washed three times with water. The green rind,accounting for ˜60% wet weight of the whole leaf, are generallydiscarded as waste by manufacturers. It contained the green rind properas well as some pulp left behind after filleting. The fibers wereisolated from them in a similar way to those from pulp followinghomogenization. They were washed extensively, at least three times, withwater, then dried and stored at RT before being used for pectinextraction.

Extraction. The chelating agent EDTA was used for extraction of Aloepectins from the cell wall fibers. The fibers were suspended in water at0.2-2% (w/v). The EDTA stock solution was prepared at 0.5 M and a pH of7.0 or 8.0 and added to the fiber suspension. The final concentration ofEDTA used was 25 mM. The final pH of the fiber suspensions was adjustedwith NaOH to the indicated values. The extraction was done with stirringat either RT or with HT, or in a sequential manner—RT extractionfollowed by HT extraction. HT was carried out up to 80° C. and thenstopped before the separation step. In the sequential extraction, theremaining fibers following the RT extraction were re-suspended to thesame volume in water without washing and fresh EDTA was added at thesame concentration as for the RT extraction (FIG. 4). Followingextraction, remaining fibers were removed by centrifugation (500 g, 15min) or by filtering with a no. 18 sieve followed by gauze spongefiltering. The gauze sponges (4×4, 8 ply) were used with three piecestogether and set up in a disc filter frame. The sponges were washed withwater before use. The gauze sponge filtration was highly efficient inremoving the residual small fibers after the sieve filtration. Whennecessary, the extract was passed through the sponge filter twice. Thefiltrate was essentially clear. For quantitative studies on yields fromsequential extraction, fibers were always removed by centrifugationfollowing the first round of extraction at RT. Alcohol (ethanol) wasadded to the clear supernatant or filtrate to a final concentration of75% (v/v). The precipitates were collected by centrifugation (500 g, 15min) or with the no. 18 sieve and washed twice with 75% alcohol. Thealcohol wash step was necessary to remove residual EDTA. Theprecipitates were then pressed to remove alcohol, dried, and stored atRT before use.

The extraction of Aloe pectins with the chelating agent EDTA was foundto be highly efficient and a yield as high as 50% (w/w) could beobtained. The pectins obtained had an average galacturonic acid contentabove 70% (w/w) (Table 1). The pH was found to have a major effect onthe pectin yield with EDTA extraction (Table 2). A 5 mg/ml fibersuspension in water had a pH of ˜3.7 (3-4). The pH of the fibersuspension was 7.7 (7.5-8.0) following addition of pH 8.0 EDTA stock toa final concentration of 25 mM. A pH of 6.4 (6.3-6.5) was obtained whena pH 7.0 EDTA stock solution was used to give a final concentration of25 mM. The pH 5.0 was obtained by using a pH 5.0 sodium acetate bufferat a final concentration of 20 mM, a common condition for pectinextraction. It was found that there was no major difference in yieldfollowing RT extraction at a pH from 5.0 to 7.7 (Table 2). A majoreffect of pH, however, was found during HT extraction. A yield increaseby >20% was noted at pH 7.7 as compared to pH 5.0 or pH 6.4 during HTextraction of fresh fibers (Table 2). Furthermore, a nearly 2-foldincrease in yield was noted when the remaining fibers from the firstround of RT extraction were extracted under HT with fresh pH 8.0 EDTAadded as compared to using pH 7.0 EDTA (Table 2). The pH values of thefiber suspensions did not change significantly at the end of RTextraction (Table 3). However, after re-suspending in water and additionof fresh EDTA, the pH (˜8.5) of the suspensions was actually higher thanthat of EDTA stock solutions (pH 8.0) (Table 3). It was further foundusing the fresh fibers under HT extraction that the pH 8.5 extractiondid give a much higher yield, more than 2-fold higher than that at pH5.0 and ˜40% higher than that at pH 7.7 (Table 4). Increasing the pH to9.0, however, did not improve the yield much further (<10%) as comparedto pH 8.5. Ensuing experiments also showed that a substantial increase(20%) in yield was also obtained with RT extraction at pH 8.5 (Table 4).

RT was less efficient than HT during extraction. The yield was similarbetween these two conditions provided the RT extraction was extended intime. The yield by RT extraction approached the maximum by ˜4 hrs.Further extension of the extraction time did not significantly improvethe yield. The yield of the second extraction with HT varied dependingon the length of the first RT extraction; therefore the yield with HTwould be higher if RT extraction was performed for only 1 hr, or lowerwhen the RT extraction was performed for 4 hrs or longer (Table 2).

Repeated extraction under the same conditions produced a progressivelylower yield. The yield decreased by approximately half with eachextraction. The remaining fibers can therefore be suspended in half thevolume from the previous extraction.

EDTA and fiber concentrations also influenced the extraction efficiency.When 25 mM EDTA was used with a 2 mg/ml fiber suspension, a yieldbetween 50-60% could be obtained with a single extraction under HT. Whenusing a 5 mg/ml fiber suspension with the same EDTA concentration, theyield decreased to ˜30%. With the sequential RT to HT extraction asshown in FIG. 4, a combined yield of 40-50% could be readily obtained.No difference in yield was noted between alcohol treated andnon-alcohol-treated fibers.

Other chelating agents were also considered for Aloe pectin extraction.Ammonium oxalate was not used because it is considered a toxic agent.Using sodium hexametaphosphate, a considerable yield was obtained;however, this agent was difficult to remove because of precipitateformation in alcohol solution and an acid (HCl or HNO₃) precipiationstep was required before the alcohol wash.

Other conditions were also examined for Aloe pectin extraction. Hotdilute acid and cold alkaline solutions are two other common conditionsfor pectin extraction. Both of them can cause extensive degradation.Commercial pectins from citrus and apple were extracted under the hotdilute acid condition. Using this condition for the Aloe pectin, the pHof fiber suspensions was adjusted to 1.5 with HCl followed by HT up to80° C. The yield obtained is much lower compared to using EDTAextraction (Table 5). The extraction by HT in water alone yieldedvirtually no alcohol precipitable materials. Renault and Thibault(Renault and Thibault, Carbohydrate Research, 1993, 244, pp. 99-114)reported that extraction of apple and sugar beet fibers in PBS (pH 6.5)with HT (80° C.) generated a high yield similar to that by EDTAextraction. Using this condition, only a low yield was obtained from theAloe vera pulp fibers (Table 5). Cold alkaline extraction was performedwith 50 mM NaOH or 50 mM Na₂CO₃ at 4° C. The pH in suspension was 11.5with 50 mM NaOH and 10.5 with 50 mM Na₂CO₃. After 1 hr at 4° C., a verylow yield was obtained with 50 mM Na₂CO₃. No alcohol precipitablematerials were obtained with 50 mM NaOH. When the extraction was done atRT for 1 hr, no yield was obtained with either agent, suggesting thatpectins are rapidly degraded under these conditions.

Together, these results showed that extraction with EDTA at pH 7.0-8.5is the most efficient extraction method for Aloe pectin. With thesequential RT to HT extraction scheme outlined in FIG. 4, a high yield(40-50%, w/w) could be obtained along with production of both HMW andLMW Aloe pectins. The uniqueness of this extraction procedure was thehigher pH (7.0-8.5) used. The reason behind this higher pH is that Aloepectins are naturally LM (see below) which are more resistant toβ-elimination under alkaline conditions and EDTA functions mostefficiently at a pH above 7.0. In addition, EDTA is more soluble at a pHabove 7.0 and can therefore be more readily removed during alcoholprecipitation and wash steps.

The green rind fibers produced a similar yield of pectin compared to thepulp fibers when extracted with the pH 8.0 EDTA (Table 6). This rindpectin was equally rich in Gal A (Table 1). The amount of fibersobtained from the rind was more than 10 times higher than that from thepulp (per unit of leaves) (Table 6). This is consistent with the factthat the rinds consisted much smaller cells as compared to the pulp(FIGS. 2 and 3). Together, these results indicated that a very largeamount of Aloe pectin can be obtained from the rind portion of the leaf,which is currently discarded as waste materials by some manufacturers.

To extract LM/HMW Aloe pectins with EDTA at about room temperature, theworkable pH range appeared to be between about 5 and about 8.5,preferably about 8-8.5. To extract LM/LMW Aloe pectin with EDTA atelevated temperature (for example at about 80° C.), the workable pHranges appeared to be between about 5 and about 8.5, preferably about8.0. At pH of higher than 6.5, EDTA extraction of HM pectins from othersources at elevated temperature would lead to the degradation of theproducts. For the extraction of pectins from other plant sources usingEDTA or other chelating agents, the reported pH ranges are 4-6.5. TABLE1 Galacturonic acid content of Aloe pectins. UA EDTA extraction contentDM*** (%, Phenol Origin of cell wall fibers* conditions** (%, w/w)mole/mole) (%, w/w) Pulp, BAM 20926 (AP BA) Slight alkaline, HT 64 — —Pulp, BAM 20926 (AP B8) Acidic, HT 89 — — Pulp, BAM 20926 (AP B8-2)Slight alkaline, HT 85 — — Pulp, BAM 10679 (AP B9) Acidic, HT 84 — —Pulp, BAM 10679 (AP B9-2) Slight alkaline, HT 86 — — Pulp, BAM 10679 (APB10) Acidic, HT 69 — — Pulp, BAM 10679 (AP B14) Acidic, HT 87 — — Pulp,BAM 10679 (AP B15) Slight alkaline, RT 81 35 0.064 Pulp, BAM 10679 (APB15-2) Slight alkaline, HT 79 40 0.036 Pulp, BAM 10679 (AP 10679) Slightalkaline, HT 77 9.4 0.05 Pulp, crude (AP B16) Slight alkaline, RT 93 1.1<0.03 Pulp, crude (AP B16-2) Slight alkaline, HT 92 17.5 <0.03 Pulp,crude (AP 97-1) Slight alkaline (RT & 91 4.4 <0.03 HT) Rind, crude (APrind B1) Slight alkaline, RT 81 4 0.045 Rind, crude (AP rind B1-2)Slight alkaline, HT 84 9.5 0.041 Rind, crude (AP rind B2) Slightalkaline, RT 75 — 0.219*The numbers following BAM (bulk acetylated mannan) are BAM lot numbers.Aloe pectin serial numbers are indicated in parenthesis and -2designates pectins obtained by HT extraction following the RTextraction.**Slight alkaline, pH 7.0-8.5; acidic, pH 5.0; RT, room temperature; HT,heating.***DM, degree of methylation.

TABLE 2 Aloe pectin yield (%, w/w) obtained under various extractionconditions. Extraction Conditions¹ pH 7.0 EDTA and pH 7.0 pH 8.0Extraction 20 mM pH 5.0 EDTA EDTA temperature² NaAc (pH5.0) (pH6.4)(pH7.7) Exp 1 HT 22 26 32.3 Exp 2 RT (1 hr) — 14.4 16 HT — 15 24.4 Totalyield — 29.4 40.4 Exp 3 RT (4 hr) 28 31.6 30 (DM = 30%)³ (DM = 29%) (DM= 19%) HT 5.6 5.8 13.5 Total yield 33.6 37.4 43.5¹The 5 mg/ml crude pulp fiber suspensions were used. Numbers in bracketsindicates the pH of the fiber suspensions after addition of EDTA.²RT, room temperature; HT, heating.³DM, degree of methylation.

TABLE 3 The pH of fiber suspensions before and extraction with EDTA.Extraction Conditions¹ pH 7.0 EDTA and 20 mM pH pH 7.0 pH 8.0 5.0 NaAcEDTA EDTA pH after addition of EDTA 5.0 6.4 7.7 pH following RTextraction 5.06 6.4 7.74 pH after re-suspending and addition 5.15 6.88.56 of fresh EDTA and before HT extraction¹The 5 mg/ml crude pulp fiber suspensions were used which had a pH of3.5.RT, room temperature;HT, heating.

TABLE 4 Further evaluation of the effect of pH on the Aloe pectin yield(%, w/w) with EDTA extraction. Extraction with heating in the presenceof EDTA¹ pH 5.0 (pH 7.0 EDTA pH 8.5 (pH 8.0 in 20 mM EDTA and pH FiberExtraction pH 5.0 pH 7.7 (pH 8.0 adjustment source temperature² NaAc)EDTA alone) with NaOH) Pulp, HT 18 32.4 44.8 crude (DM = 27%)³ (DM =29%) (DM = 30%) Rind, RT 26 26 32 crude (DM = <10%) (DM = <10%) (DM =<10%)¹The 5 mg/ml fiber suspensions were used. DM, degree of methylation.²RT, room temperature; HT, heating.³DM, degree of methylation.

TABLE 5 Aloe pectin yields obtained with non-EDTA-based extraction.Extraction Conditions¹ 50 mM 50 mM Heating at pH 1.5 Heating at pHNa₂CO₃ Na₂CO₃ Heating in (adjusted with 6.5 (pH 6.5 (pH 10.5) at (pH10.5) at RT water HCI) PBS) 4° C. for 1 hr. for 1 hr. Yield 0 9.6 10.67.5 0 (%, w/w)¹The 5 mg/ml crude pulp fiber suspensions were used.

TABLE 6 Extraction of Aloe pectin from green rind fibers. Fresh AloeVera Leaves Pulp Rind Wet weight after separation 188 g (33%) 376 g(67%) Fibers obtained after 0.34 g 5.23 g homogenization, 18# sievefiltration, and washing Pectin yield * EDTA-RT (1^(st) round) 10.8%(w/w) 17.5% (w/w) EDTA-HT (2^(nd) round) 26.4% (w/w) 25.5% (w/w) Total37.2% (w/w)   43% (w/w) Pectin powder color White-off white Lightgreen-brown* The fibers were extracted at 5 mg/ml and RT extraction was performedfor 1 hr.

EXAMPLE 4 Pectin Purification by Ion Exchange Chromatography

The ion exchange chromatography was performed on a Pharmacia BiotechAKTA explorer chromatography system. The column was three PharmaciaHi-trap Q, 5 ml cartridges connected in series. Aloe pectins weredissolved in water at 1 mg/ml and loaded onto the column at a flow rateof 1 ml/min. After washing with 15 ml of water, bound materials wereeluted with a linear gradient of NaCl (0-1.0 M). The column eluant wasmonitored by UV absorbance at 215, 254, and 280 nm. Fractions containingpectin formed precipitates which were collected by low speedcentrifugation, pooled, and redissolved in water. They were thendesalted by passing through a Sephadex G-25 column. Thepectin-containing fractions were collected, dried, and stored at roomtemperature.

EXAMPLE 5 Uronic Acid Assay

The m-hydroxyldiphenyl-based uronic acid assay was carried out asdescribed by (Blumenkratz and Asboe-Hansen (1973), AnalyticalBiochemistry 54, pp. 484-489). Briefly, samples or standards in 200 μlpyrogen-free water were mixed with 1.2 ml concentrated H₂SO₄ containing0.0125 M sodium tetraborate and then immediately put on ice. The sampleswere then kept in boiling water for 5 min followed by cooling in awater-ice bath. 20 /Al of 0.15% (w/v) m-hydroxyldiphenyl in 0.5% NaOHwas then added to each reaction. After mixing, the samples were kept atroom temperature for 30 min. The absorbance at 520 nm was thendetermined. Gal A was used to generate a standard curve (0, 1, 2, 4, 6,8, and 10 μg). Mannose was used as a neutral sugar control. All sampleswere tested at 20 μg or less.

The average Gal A content of different Aloe pectins was above 70% (Table1). There were no significant differences between the Gal A contents ofpectins extracted under different conditions.

EXAMPLE 6 Sugar Composition and Linkage Analysis

Fluorophore-Assisted Carbohydrate Electrophoresis (“FACE”) is a fast andsimple technique for sugar composition analysis. It allowed for initialexamination and comparison of various samples and was carried outaccording to the procedure provided with the FACE sugar compositionanalysis kit (Glyco, Inc.). Briefly, polysaccharides were hydrolyzedwith 2N trifluoroacetic acid (TFA) at 100° C. for 5 hrs and then labeledwith a fluorescent dye (AMAC, 2-aminoacridone) and electrophoresed.Carbohydrate bands were visualized under a UV light (Fotodyne 3-3000).Besides the neutral sugar standards provided in the kit, Gal A and Glc Awere also used.

Composition analysis by TMS derivatization. Samples were subjected topreliminary aqueous hydrolysis in 2N TFA for 6 hrs at 105° C. TFA wasremoved by evaporation under nitrogen and the partially hydrolyzedcarbohydrate residue was subjected to methanolysis in 2M methanolic HClfor 16 hrs at 80° C. to complete the hydrolysis with the formation ofmethyl glycosides. Methanolic HCl was removed under nitrogen and themethyl glycosides were subjected to N-acetylation inmethanol-pyridine-acetic anhydride for 6 hrs at room temperature. Thesolvents were evaporated and the residues were trimethylsilylated usingTri-Sil at 80° C. for 20 min. The resulting TMS-methylglycosides, wereanalyzed by GC-MS using a 30 m DB-5 capillary column equipped with amass selective detector.

Linkage analysis. The Hakomori method (Hakomori, Journal ofBiochemistry, 1984, 55, pp. 205-212) of methylation with superdeuteridereduction was used. The samples were suspended in DMSO and subjected tosonication at 60° C. for 36 hrs in a bath type ultrasonicator. Sampleswere then methylated using potassium methylsulfonyl carbanion (3.6 M)followed by the addition of a 50-100 fold excess of methyl iodide. Thepartially methylated material was isolated by reverse phase cartridgechromatography and subjected to carboxyl reduction. The samples werethen purified and subjected to remethylation according to the Hakomoriprocedure. The sample was then hydrolyzed and converted to partiallymethylated alditol acetates. The resulting PMAA derivatives wereanalyzed by GC-MS using a 30 m SP-2300 capillary column.

Sugar composition analysis using FACE showed that the extracted pectinwas richer in Gal A as compared to the cell wall fibers. The detailedcompositions were obtained with TMS derivatization and GC-MS analysis.In Table 7, the sugar compositions of three samples, AP 10679, AP 10679(purified as described in Example 4), and AP97-1, are presented (Seealso Table 1). Sample AP 10679 was obtained by HT extraction fromalcohol-treated fibers as described in Example 3. Sample AP97-1 was atrial production sample extracted from non-alcohol-treated crude fibers.The fibers were extracted twice at room temperature followed by HTextraction. The pectins obtained from the two extraction conditions werecombined and the ratio of the pectins extracted at RT over those by HTextraction was ˜2:1. TABLE 7 Sugar composition (*, mole/mole) of Aloepectins. AP 10679 AP 10679 (purified) AP 97-1 (5)¹ (5) (5) Ara 4.2 1.84.0 Rha 11.1 4.4 10.3 3-Me-Rha 0.8 0.5 0.8 Fuc 0.6 0.4 0.6 Xyl 3.9 1.22.4 Man 1.6 0.3 3.5 Gal 8.5 6.8 14.8 Glc 1.1 0.7 0.4 Gal A 67.5 83 63.2DM LM (9.4) LM LM (4.4) (natural) (natural) (natural) DAc 9.0 ≦2.8 9.1Total phenol (%, w/w) 0.058 — <0.03 Rha/Gal A 0.16 0.05 0.16 Gal/Gal A0.13 0.08 0.23¹The number in parenthesis is the reference number. See the referencelist at the end of Table 15.

The sugar composition analysis showed that Gal A was the primary sugar,67% in AP 10679 and 63.2% in AP 97-1. The rhamnose and galactose are themost abundant neutral sugars, accounting for 10-11.1% and 8.5-14.8%,respectively. Among the minor neutral sugars, a modified sugar,3-OMe-rhanmose was detected, which accounted for about 10% of totalrhamnose. The sugar compositions were very similar between the twosamples, except for a small amount (<0.5%) of GalNAc and glyceroldetected in AP 10679.

The purified AP 10679 showed an enriched Gal A content and a reducedneutral sugar content, suggesting that some of the neutral sugarsdetected in the unpurified sample may not be associated with the pectin.The rhamnose and galactose were still the most abundant neutral sugars.The 3-OMe-rhamnose was also still present, again accounting for ˜10% oftotal rhamnose. TABLE 8 Glycosyl linkage in AP 10679. % TotalMonsaccharide Linkage area¹ Area ratio Arabinose Terminal (fur) 7.2 0.39Terminal (pyr) 0.6 0.03 5-linked (fur) 1.1 0.06 2-linked (pyr) 0.7 0.042,3-linked (pyr) 0.6 0.03 Rhanmose Terminal 4.0 0.22 2-linked 14.7 0.83-linked 2.2 0.12 2,3-linked 0.7 0.04 2,4-linked 6.3 0.34 XyloseTerminal 5.5 0.3 4-linked 4.8 0.26 2,4-linked 1.2 0.07 Fucose Terminal3.9 0.21 3,4-linked 1 0.05 Mannose Terminal 1.2 0.07 4-linked 4 0.22Galactose Terminal 5.7 0.31 3,4-linked 1.4 0.06 3,6-linked 0.6 0.034,6-linked 0.5 0.03 Glucose 4-linked 2.5 0.14 Galacturonic acid Terminal2.8 0.15 4-linked 18.3 1 Galacturonic acid Terminal 2.6 0.14 2-linked2.3 0.13 2,4-linked (Gal UA/GlcUA) 0.9 0.05¹Percent of total area is normalized to 1-4 linked galacturonic acid.

TABLE 9 Glycosyl linkage in AP97-1. % total Monsaccharide Linkage area¹Area ratio Arabinose Terminal (fur) 6.2 0.34 Terminal (pyr) 0 0 5-linked(fur) 1.6 0.09 2-linked (pyr) 0.8 0.04 2,3-linked (pyr) 0.7 0.04Rhanmose Terminal 3.8 0.21 2-linked 11.1 0.61 3-linked 1.5 0.082,3-linked 0.6 0.03 2,4-linked 10.7 0.58 Xylose Terminal 6.9 0.384-linked 2.9 0.16 2,4-linked 1.1 0.06 Fucose Terminal 3.3 0.183,4-linked 0.8 0.04 Mannose Terminal 2.7 0.15 4-linked 8.2 0.45Galactose Terminal 5.3 0.29 3,4-linked 1.5 0.08 3,6-linked 0.5 0.034,6-linked 0.6 0.03 Glucose 4-linked 1.7 0.09 Galacturonic acid Terminal2.6 0.14 4-linked 18.3 1 Galacturonic acid Terminal 2.3 0.12 2-linked2.5 0.14 2,4-linked (Gal UA/GlcUA) 0.9 0.05¹Percent of total area is normalized to 1-4 linked galacturonic acid.

The sugar linkage data on AP 10679 and AP 97-1 are shown in Tables 8 and9. The major linkages detected were 1, 4 linked Gal A and 1, 2 linkedrhamnose. The 1, 4 linkage for Gal A is the same as for other pectins.No other linkage was detected for Gal A (Tables 8 and 9). Besides the 1,2 linkage, rhamnose residues were also 1, 2, 4 linked with a smallportion (0.6 or 0.7%) 1, 2, 3 linked. The rhamnose with the 1, 2 and 1,2, 4 linkages accounted for the major portion of the total rhamnoseresidues, suggesting that most of the rhamnose residues detected are inthe pectin backbone. Since the 1,2,4 linked rhamnose was much more thanthe 1,2,3 linked in both samples, the neutral sugar side chains aretherefore most likely linked to the backbone at the O-4 position ofrhamnose residues. TABLE 10 The sugar composition (%, mole/mole) of Aloepectin in comparison with commercial pectins (unpurified). AP Sugar10679 AP 97-1 Apple Apple K Apple U Lemon A Lemon B Citrus Citrus beet(5)¹ (5) (1) (9) (9) (4) (4) (8) (2) (6) Ara 4.2 4.0 1.4 7.23 3.42 2.92.7 1.44 3.3 13.2 Rha 11.1 10.3 2.9 2.03 1.83 1.8 1.4 1.74 1 3.23-Me-Rha 0.8 0.8 — — — — — — — — Fuc 0.6 0.6 — — — — — — — — Xyl 3.9 2.42.2 1.24 0.46 0.17 0.16 0.16 0.1 0.3 Man 1.6 3.5 tr 0.11 0.11 0.17 0.160.21 0.2 0.3 Gal 8.5 14.8 3.4 9.6 7.43 6.0 6.7 5.41 4.8 7.1 Glc 1.1 0.44.7 18.87 8.57 0.5 0.87 0.89 0.6 0.4 Gal A 67.5 63.2 8 64 76.68 88 8890.2 90 58.8 DM LM LM LM LM HM HM HM — HM HM (9.4) (4.4) (28) (42)(73.6) (71.5) (72) (71.4) (66.6) (natural) (natural) DAc 9 9.1 — — 1.41.6 — <1 25.4 Rha/Gal A 0.16 0.16 0.034 0.004 0.015 0.02 0.016 0.0160.011 0.054 Gal/Gal A 0.13 0.23 0.04 0.15 0.1 0.068 0.076 0.06 0.0530.12 Powder White/off white Tanned Light yellow/brown Tanned colorSolution Clear Cloudy Cloudy clearness¹Reference number. See the reference list at the end of Table 15.

TABLE 11 The sugar composition (%, mole/mole) of purified Aloe pectin incomparison with other purified pectins. AP 10679 Sugar Sugar Sugar(purified)/ Citrus/ Citrus/ Apple/ Apple/ Apple/ Apple/ Beet/ Beet/Beet/ Chela¹ Acid³ Acid Chela Acid Acid Chela Acid Chela ChSS (5)² (15)(2) (11) (9) (9) (12) (13) (13) (11) Ara 1.8 1.9 1.8 13 2.77 2.1 4 9.82.9 16 Rha 4.4 0.6 0.7 2 1.16 0.58 1 3.1 1.1 2 (+Fuc) (+Fuc) 3-Me- 0.5 —— — — — — — — — Rha Fuc 0.4 — — — — — — — — tr Xyl 1.2 0.2 0.1 1 1.040.35 1 0.3 0.3 tr Man 0.3 0.1 — — 0.11 — tr 0.06 0.02 — Gal 6.8 2.7 3.23 5.2 5.02 3 4 3.3 6 Glc 0.7 0.2 0.2 1 2.08 1.75 1 0.2 0.3 tr Gal A 8366.3 94 88 87.38 89.95 90 82 92 76 DM LM HM HM — HM HM — HM HM —(natural) (79.1) (72) (75.8) (72.3) (62) (60) DAc ≦2.8 2 <1 — — — — 3515 — Rha/Gal A 0.053 0.009 0.007 0.022 0.013 0.006 0.011 (0.038) (0.012)0.026 Gal/Gal A 0.08 0.04 0.034 0.034 0.06 0.06 0.033 0.0 0.036 0.078¹Chela, extracted with chelating agent.²Acid, extracted under the acidic condition.³Reference number. See the reference list at the end of Table 15.

TABLE 12 The sugar composition (%, mole/mole) of Aloe pectins incomparison with others extracted with chelating agents. Sugar Sugar AP10679 AP 97-1 Apple Apple Apple Citrus Rapeseed Sunflower Beet BeetPotato (5)¹ (5) (14) (12) (11) (10) (3) (8) (12) (15) (7) Ara 4.2 4.0 2815 4.9 4 27 0.75 25 15 2.8 Rha 11.1 10.3 3 2.9 1.6 1 2.0 1.77 2.5 2.22.2 (+fuc) 3-Me-Rha 0.8 0.8 — — — — — — — — — Fuc 0.6 0.6 0.01 0.1 — —0.6 — — — 0.3 Xyl 3.9 2.4 4 3.9 1.4 <1 8.2 0.31 — 0.6 0.5 Man 1.6 3.5 1— tr 1 3.0 0.1 — 0.3 1.3 Gal 8.5 14.8 8 8.4 6.5 2 9.2 0.68 7.7 5 14.6Glc 1.1 0.4 1 1.2 3.9 2 3.2 1.18 — 0.7 0.9 Gal A 67.5 63.2 55 67.4 81 9047 95.6 64.1 76 77.3 DM LM (9.4) LM (4.4) HM HM HM HM — LM (38.5) HM HM˜50% (natural) (natural) (82) (75) (60) (79) (natural) (58) (64) DAc 9.09.1 13 12 2 2 — 2.01 33 20.6 — Rha/Gal A 0.16 0.16 0.055 0.043 0.0190.011 0.045 0.008 0.039 0.029 0.028 Gal/Gal A 0.13 0.23 0.15 0.12 0.080.02 0.19 0.01 0.12 0.066 0.189¹Reference number. See the reference list on the next page.

REFERENCES FOR TABLES 7, 10, 11 AND 12

-   1. Axelos, M. A. V. and Thibault, J. F. (1991), Int. J. Biol.    Macromol, 13:77-82.-   2. Axelos, M. A. V. and Thibault, J. F. (1989), Int. J. Biol.    Macromol, 11;186-191.-   3. Eriksson, I. Andersson R., and Aman, P. (1997), Carbo. Res.,    301:177-185.-   4. Kravtchenko, T. P., Voragen, A. G. J., and Pilnik, W. (1992),    Carbo. Polymers, 18:17-23.-   5. Analysis reports on Aloe Pectins from Complex Carbohydrate    Research Center at University of Georgia.-   6. Guillon, F. and Thibault, J. F., (1990), Carbo. Polymers,    12:353-374.-   7. Jarvis, M. C., Hall, M. A., Threlfall, D. R.,m and Friend, Jr.    (1981), Planta. 152:93-100.-   8. Miyamoto, A. and Chang, K. C., (1992), J. Food Sci.,    57:1439-1443.-   9. Pilnik, W. (1981), APRIA symposium on fiber in human    nutrition, p. 91, Paris.-   10. Ros, J. M., Schols, H. A., and Voragen, A. G. J. 1996), Carbo.    Res. 282:271-0284.-   11. Renard, C. M. G. C., Voragen, A G. J., Thibault, J. F., and    Pilnik, W. (1990), Carbo Polymers 12:9-25.-   12. Renard, C. M. G. C. and Thibault, J. F. 1993), Carbo REs.,    244:99-114.-   13. Toumbouts and Thibault (1986), Carbo Res. 154:177-187.-   14. Schols et al., (1995), Carbo Res., 275:343-360.-   15. THibault, J. F. (1988), Carbo Polymers, 8:209-223.-   16. Thibault, J. F.,.Dreu, R. D., Geraeds, C. C. J., and    Rombounts, F. M. (1988), Carbo. Polymers, 9:119-131.

The linkage experiments also detected Glc A which was not detected inthe composition experiments because its peaks overlapped those of themuch stronger Gal A peak (Tables 8-9). Among other sugars, galactose waseither 1, 3, 4 or terminally linked with a small portion 1, 3, 6 or 1,4, 6 linked, arabinose(fur) either 1,5 or terminally linked, arabinose(pyr) 1, 2 linked, fucose 1, 3, 4 linked, xylose 1, 4 linked, mannosemainly 1, 4 linked, glucose 1, 4 linked, and Glc A 1, 2 linked. The 1, 4linked mannose is similar in linkage to the mannan found in liquid gelinside pulp mesophyll cells. Thus, presence of 1, 4 linked mannose couldbe the result of residual liquid gel still associated with the mesophyllcell wall fibers.

EXAMPLE 7 Acidic Polysaccharide Gel Electrophoresis

The gel electrophoresis for separation of acidic polysaccharides wascarried out as described by Misevic (Misevic, Methods in Enzymology,1989,179, pp.95-110) using the Bio-Rad minigel apparatus. Tris-boricacid (pH 9.0) was used as both the gel and running buffer. A 15%polyacrylamide gel was found to be optimal. The gels were stained withalcian blue and destained with 2% (v/v) acetic acid.

EXAMPLE 8 Enzyme Digestion of Aloe Pectin

Aloe pectin was dissolved in 50 mM sodium acetate buffer (pH 5.0).Endo-polygalacturonase (EC3.2.1.15) was added at various concentrations(0.25-25 mU). After incubation at room temperature for 1 hr, sampleswere immediately mixed with the sample buffer (pH 9.0) and separated byacidic polysaccharide gel electrophoresis as described above.

Endo-polygalacturonase is specific for α1-4 linked Gal A residues inpectins. The results showed that this enzyme degraded Aloe pectin in adose-dependent manner; the higher the enzyme concentration, the smallerthe size of remaining pectin molecules as demonstrated by fastermigration in the gel. No effect was observed on heparin, a non-pectincontrol. This result confirmed the 1-4 linkage between Gal A residues inAloe pectin and also indicated that the configuration of this linkage isα.

EXAMPLE 9 Acetylation, Methylation, and Total Phenol

Acetylation and Methylation

The acetyl groups were detected by derivatization with hydroxylamine HCland ferric acid. Acetylcholine (0.001-0.0005 N) was used as thestandard. Samples were tested at various concentrations (0.2-0.8 mg/ml).Both samples and standards were prepared in 1 ml 0.001 N acetate bufferand mixed with 2 ml of hydroxylamine HCl reagent (2 M hydroxylamine HClmixed 1:1 with 3.5 N NaOH). After about 1 min, 1 ml 4 N HCl was added.After another 1 minute, 1 ml of 0.37 M ferric chloride (in 0.1 N HCl)was added. Following mixing, the absorbance at 540 nm was measured. Theamount of acetyl groups in mole was determined by extrapolating againstthe linear regression curve of the standard. The degree of acetylation(DAc) of the pectins was expressed as % (mole/mole) of Gal A.

Degree of methylation (DM) was determined using the selective reductionmethod described by Maness (Maness, Analytical Biochemistry, 1990, 185,pp. 346-352) with modifications. Pectin samples were prepared in 1 Mimidazole-HCl buffer (pH 7.0). For each test, 1 mg sample in 400 μl wasused. NaBH₄ (40 mg) was added and the sample was kept on ice for 1 hr toselectively reduce the methyl-esterified Gal A residues. Then, 0.1 mlacetic acid was added to remove the remaining NaBH₄. The sample wasdiluted with 0.5 ml water and the pectins were precipitated with 4volumes of 95% ethanol. After being dried, the sample was dissolved inwater and the Gal A content was determined as described above. Thecontrol went through the same steps except for the addition of NaBH₄.The DM was determined by the following formula: DM=[(moles of Gal A inthe control-moles of Gal A in the reduced)/moles of Gal A in thecontrol]×100.

It was found that the alcohol precipitation and drying steps could beeliminated without affecting the results. Thus, after addition of aceticacid, 9.5 ml of water was added, giving a pectin concentration of 0.1mg/ml or 20 μg/200 μl—the upper limit for the uronic acid assay.

Determination of Total Phenol

The method described by Rombouts and Thibault (Carbohydrate Research,1986,282, pp. 271-284) was used. Pectin samples in 0.6 ml water weremixed with 0.6 ml of Folin-Ciocalteu reagent. After 3 min, 0.6 ml of 1 Msodium carbonate was added. The mixtures were left at RT for 1 hr beforethe 750 nm absorbance values were determined. Ferulic acid was used as astandard. Some precipitates formed after pectin samples were mixed withthe reagents. They appeared to be colorless and removed bycentrifugation at 3,000 rpm for 15 min before absorbance measurement at750 nm.

Methylation, Acetylation, and Total Phenol

The results obtained with the selective reduction method showed thatAloe pectins had a DM below 40% and often <10% (Table 1). The DMs of twopectin samples (citrus LM and citrus HM) from Sigma Chemical Co. weredetermined by this method to be 24% (±3.5) and 58% (±3.5), beingconsistent with the values (28% and 64%, respectively) provided by thesupplier.

It was found that Aloe pectins obtained by RT extraction had a DM lowerby 5-10% as compared to those obtained by HT extraction (Table 1). Itwas also found that RT extraction at pH 7.7 produced pectin with a DMlower by 10% as compared those by RT extraction at pH 5.0 or 6.4 (Table2). This latter observation appeared to be consistent with the fact thatincreasing pH at RT favors the demethylation reaction over theβ-elimination. No difference was noted when HT extraction was performedat various pH (5.0-8.5) (Table 4).

The rind pectin was also LM (Table 1). In fact, they consistentlyexhibited a DM below 10% (Tables 1 & 4). This suggests that the rindpectins may naturally have an even lower DM as compared to those of pulppectins.

Acetylation was detected with a chemical method as described above. AP10679 and AP 97-1 exhibited a DAc of 9.0% and 9.1%, respectively.However, the DAc of the purified AP 10679 was found to be ≦2.8%. Thissuggests that Aloe pectin also has a low level of acetylation (Table 7).

Aloe pectins had a very small amount of phenols (0-0.22%, w/w) (Table1).

EXAMPLE 10 Molecular Weight Determination By Size ExclusionChromatography (SEC)

The SEC was performed using TSK-Gel G5000 PWXL column (Toso Haas).Samples were prepared at 0.3 mg/ml in water with 0.05% (w/v) sodiumazide. 50 il of the sample was injected and eluted with 0.05% sodiumazide at 1 ml/min. Refractive index was measured in line. Pullulans(4.04×10⁵, 7.88×10⁵, and 1.66×10⁵ Da),were used as standards. Themolecular weight was calculated against the linear regression line ofthe standards.

Aloe pectins generally exhibited only one major peak. This is consistentwith findings on other pectins. Aloe pectins obtained by RT extractionhad an average molecular weight of 1.1×10⁶ (0.785-1.36×10⁶ Da), whichwas ˜5 times larger than the average size 1.9×10⁵ (0.375-6.08×10⁵ Da) ofthose obtained by HT extraction. Pectins extracted with HT fromremaining fibers of RT extraction had a similar molecular size to thoseextracted with HT from fresh fibers.

The sizes of pectins were also analyzed by acidic polysaccharide gelelectrophoresis. Profiles obtained from gel electrophoresis wereconsistent with the results obtained by size exclusion chromatography,i. e., the sizes of pectins obtained by RT extraction were much larger(migrated much slower in gel) than those by HT extraction. Using thistechnique, it was also observed that pectins extracted with HT at low pH(5.0) had a comparable size to those obtained by RT extraction. Thissuggests that pH is the most important factor in determining the size ofpectins obtained, although heating is also important. These findings areconsistent with the general properties of pectins, i.e., they are moststable at low pH (3-4) and low temperature.

Together, the pectins obtained by RT extraction or HT extraction at lowpH (5.0) are grouped as high-molecular-weight (HMW) pectins and thoseobtained by HT extraction at pH 7.0 or above are grouped aslow-molecular-weight (LMW) pectins. Thus, two classes of Aloe pectinsdistinguished by size can be readily obtained by changing extractiontemperature. This could be best achieved by following the sequentialextraction scheme outlined in FIG. 4.

The average size of HMW Aloe pectins (1.1×10⁶ Da) is much larger thanthat (0.7-1.4×10⁵ Da) of commercial pectins, which is close to that ofLMW Aloe pectins. To confirm this size difference, three commercialcitrus pectin samples, one LM (P-9311, lot 74H1092; Sigma Chemical Co.)and two HM (P-9436, lot 96H0788; Sigma Chemical Co. and PE100, lotJR071, Spectrum Chemical Co.), were analyzed by SEC under the sameconditions. Their sizes ranged from 2.0-4.6×10⁵ Da, being much lowerthan those of HMW Aloe pectins. The sizes of citrus pectins are usuallylarger than those of apple pectins (Pilnik and Voragen, Advances inPlant Biochemistry and Biotechnology, 1992, 99, pp. 219-270).

EXAMPLE 11 Viscosity

Intrinsic viscosities were determined using the Ubbelhode viscometer(No. 2). Pectins were dissolved in 0.1 M NaC1 at a concentration of0.005-0.2% (w/v) (Owens, Journal of the American Chemical Society, 1946,68, pp. 1628-1632; Axelos, International Journal of BiologicalMacromolecules, 1989, 11, pp. 186-191.) The intrinsic viscosity (η) wascalculated using double Huggins (η_(sp)/c=η+k¹η²c) and Kraemer ([lnη_(rel)]/c=η+k″η²c) extrapolation (to zero concentration) (Axelos andThilbault, International Journal of Biological Macromolecules, 1989, 11,pp. 186-191; Doublier and Cuvelier, Carbohydrates in Food, ed. A. C.Eliasson, Marcel Dekker, New York, 1996, pp. 283-318). The averages ofthe numbers obtained with these two equations are presented in Table 13in comparison with MW.

The highest intrinsic viscosity (978 ml/g) was found with a rind pectinobtained by RT extraction. The intrinsic viscosities of HMW Aloe pectinswere generally higher than those of LMW ones. The intrinsic viscositiesof HMW Aloe pectins were also generally higher than those of thecommercial citrus and apple pectins tested here. This is also consistentwith the differences in molecular weight between HMW Aloe pectins andcommercial pectins. TABLE 13 Intrinsic viscosities of Aloe pectins.Intrinsic Viscosity Pectins Fiber source Size (η, ml/g) AP 97-1 Pulp,crude 1.36 × 10⁶ (HMW) 740 AP 10679 Pulp, BAM 3.75 × 10⁴ (LMW) 68 AP B15Pulp, BAM 7.87 × 10⁵ (HMW) 262 AP B15-2 Pulp, BAM 6.45 × 10⁴ (LMW) 68 APB16 Pulp, crude 1.06 × 10⁶ (HMW) 550 AP B16-2 Pulp, crude 6.08 × 10⁵(LMW) 337 AP rind B1 Rind, crude ND* 978 AP rind B1-2 Rind, crude ND 523AP rind B2 Rind, crude ND 846 Sigma LM citrus — 2.18 × 10⁵ 51 Sigma HMcitrus — 2.02 × 10⁵ 178 Spectrum HM citrus — 4.56 × 10⁵ 297 HF HM citrus— ND 201 HF HM apple — ND 277*Not determined.

EXAMPLE 12 Calcium Gel Formation

Aloe pectins at various concentrations in water were mixed with calciumchloride solution at various concentrations along with commercial LM andHM pectins. After standing at RT for up to 24 hrs, the tubes wereinverted. If the sample flowed easily, it was considered that no gelformation occurred. If the sample did not flow or deform under its ownweight, gel formation had occurred. If the sample did not flow, butdeformed (i.e., the surface did not keep a straight line perpendicularto the side of the tube when tubes were held at a horizontal position),the system was considered as a soft gel. The results showed that Aloepectin obtained by either RT or HT extraction from either pulp or rindfibers formed firm gels in the presence of calcium as did the LM citruspectin and polygalacturonic acid (Table 14). Under the same conditions,the HM citrus pectin did not form gels. This is consistent with the factthat the Aloe pectin is a LM pectin. Pectins from citrus and apple arenaturally HM pectins. LM pectins are obtained by demethylating the HMpectins. Since no harsh conditions were applied during the extraction ofAloe pectins, especially with RT extraction, the Aloe pectin is anatural LM pectin.

With a 0.2% Aloe pectin solution, the minimum concentration of calciumchloride required for gel formation was determined to be 1-2 mM (50-100mg CaCl₂/g pectin). With increasing concentrations of pectin and/orcalcium chloride, the gel became gradually firmer. It was noted that theHMW Aloe pectins formed gels more readily than LMW Aloe pectins in thatit took less time for gels formation and the gel seemed firmer.

Increasing the ionic strength facilitated the calcium gel formation. Thespeed of gel formation gradually increased with increasing NaClconcentrations (0-0.2 M) after the addition of a fixed amount of calciumchloride.

EXAMPLE 13 Monovalent Cation-Based Gel Formation

Aloe pectins were dissolved in water at various concentrations. Thepectin solutions were mixed at RT with equal volumes of 0.3 M NaCl (2×saline), 0.3 M NaCl and 40 mM sodium acetate (pH 5.0), or 2× PBS (pH7.4). The final volumes were 1 or 2 ml. The tubes (12×75 mm) were thenplaced in a fridge at 4° C. or on ice (0° C.). The gel formation wasjudged as described in Example 12. The tubes were then returned to RT todetermine if the gel reverted back to solution. Various NaClconcentrations (0.05-1 M) were tested for gel formation. The potassiumsalt (KCl) was also tested. The salt and pectin solutions were alwaysmixed in equal volumes (1:1). For determining the effect ofendo-polygalacturonase on the gel formation, pH 5.0 acetate buffer wasadded to pectin solutions to a final concentration of 20 mM. The enzymewas then added at indicated concentrations. After standing at RT for 30min, the solutions were mixed with equal volumes of 0.3 M NaCl and thenplaced on ice. The gel formation was examined as above. When an Aloepectin solution in 0.15 M NaCl (physiological saline) was cooled to 4°C., a gel was obtained. The gel was firm and free standing when kept at4° C. just as the calcium gel; it turned quickly back to solution whenbrought to RT (22° C.). This reversible solution-gel transition could berepeated many times by changing the temperature.

Unlike the gel formation in the presence of calcium which occurredefficiently with both HMW and LMW Aloe pectins, the monovalentcation-based gel formation only occurred efficiently with HMW Aloepectins obtained from either pulp or rind fibers. The sample AP 97-1 andsimilar ones, which had molecular weights of >1×10⁶ Da, could producefirm gels at concentrations as low as 1 mg/ml in the presence of 0.15 MNaCl. Such gels were also clear when the pectin concentrations were 5mg/ml or less. With higher pectin concentrations (>5 mg/ml), gels werefirmer and slightly cloudy. With a 1 ml volume, a gel could form in ˜15min after the tube was placed on ice and returned to solution in aboutthe same time after it was brought back to RT. The gel, however, did notrevert back to solution at a temperature as high as 15° C. The gel couldform at pH 5.0 (in saline with 20 mM pH 5.0 sodium acetate) as well aspH 7.4 (in PBS).

The LMW (0.375-6.08×10⁵ Da) Aloe pectins only formed such gels at higherconcentrations (≧5 mg/ml). At 1 mg/ml, only soft gels could be obtainedwith some of the LMW samples in 0.15 M NaCl. The smallest Aloe pectinsample (0.375×10⁵ Da) formed no gel at 1 mg/ml in 0.15 M NaCl. A softgel was only obtained with this sample at a pectin concentration of 10mg/ml in 0.2 M NaCl. This suggests that the efficiency of the monovalentcation-based gel formation is dependent on the size of the pectinmolecules. As shown in Example 8, Aloe pectin could be degraded byendo-polygalacturonase. Thus, 300 μl of 2 mg/ml AP97-1 pectin solutionin 20 mM pH 5.0 sodium acetate was digested with this enzyme at variousconcentrations before mixing with an equal volume of 0.3 M NaCl andplaced on ice. The results showed that the control (no enzyme added)formed a gel and the sample with the highest enzyme concentrationremained a solution (Table 15). Between the control and the highestenzyme concentration, the transition from solution to gel was evident,i.e., the gel became softer with an increase in the enzyme concentrationuntil a complete solution was obtained at the highest enzymeconcentration. This result indicates that the size of the Aloe pectinmolecules is an important factor in monovalent cation-based gelformation.

The gel formation was also dependent on the NaCl concentration. In 0.1 MNaCl, only soft gels were obtained with samples like AP 97-1. The firmgels only formed in 0.15 M and 0.2 M NaCl. Whereas the gel formed at0.15 M NaCl was fully reversible when the gel was brought back to RT,the gel formed at 0.2 M NaCl was not readily reversible, especially forthe HMW Aloe pectins. After standing at RT for 1 hr or longer, syneresisoften occurred with the gel formed at 0.2 M NaCl, i.e., the liquid wasseparated from the gel. With higher NaCl concentrations (>0.4 M),precipitates formed at RT. The precipitates were white and amorphous athigh NaCl concentrations (0.6-1 M) and appeared to be fine granules at0.4 M NaCl.

Such cold gelation is also sensitive to the species of monovalentcations used. With KCl (0.05-1 M), no cold gel formation occurred,although precipitates were formed at higher KCl concentrations (>0.4 M)at RT.

Precipitation of pectins at high salt concentrations and RT has beenpreviously observed. However, such a reversible monovalent cation(NaCl)-based cold gelation under the physiological condition (0.15 MNaCl, pH 7.4) has not previously been described with any other pectins.So far, no such gelling system has been identified with any otherpolymers or substances in literature. Using the commercialpolygalacturonic acid, LM and HM pectins, no such monovalent coldgelation was obtained.

EXAMPLE 14 Use of Aloe Pectin as An Encapsulating Agent for ControlledRelease

The APase and APase-antibody (APase-Ab) conjugate were used forencapsulation. They were chosen because the release activity can bedirectly measured using the APase substrate pNPP. Aloe pectins at 10 or15 mg/ml in water were mixed with APase or APase-Ab at a finalconcentration of 10-20 μg/ml. The mixture at RT was dripped over about30 minutes into a 200 mM CaCl₂ bath to make beads ˜1 mm in diameter.Beads isolated by decantation were washed and kept in water at 4° C.First, spontaneous release was examined in relation to pectinconcentration and the size of pectin molecules. For release experiments,the same numbers of beads (3-5) were incubated at room temperature in100 μL of water, saline (150 mM NaCl), TN buffer, or buffers withoutNaCl at various pH for 2 hrs. The pH 3-5 was achieved with 10 mM sodiumacetate buffer and the pH 6-8 was achieved with Tris buffer. At the endof the incubation, 10 μl of the incubation media was removed and mixedwith 100 ttl of the APase substrate (pNPP). After 15 min, the reactionwas stopped with 50 μl 2M NaOH and the absorbance at 405 nm wasmeasured.

The results showed that a pectin concentration above 10 mg/ml couldefficiently inhibit the spontaneous release and pectins with largersizes entrap the target agent more efficiently (FIGS. 5 a and 5 b). Theconditions for triggering release were then examined. It was found thatthe entrapped enzymes were only released in saline (150 mM NaCl) or at apH of 7.0 or above (FIG. 5 c). The combination of these two conditionsas represented by TN buffer (25 mM Tris, 150 mM NaCl, pH 7.4) gave themost efficient release (FIG. 5 c).

Although the protein molecules used in the present experiments are largeones (APase, 140 kDa; APase-IgG, ˜350 kDa), these results clearlyindicate that there is a release mechanism in the Aloe pectin-calciumgel controlled by the salt concentration and pH. Thus, the physiologicalcondition (150 mM NaCl and pH7.0-7.4) should initiate the release oncethe beads are delivered in vivo, whereas under the storage conditions noor only minimal release occurs. This Aloe pectin-calcium gelencapsulating system should be suitable for protein molecules such asantibodies and vaccines. TABLE 14 Gel formation and degree ofmethylation (DM) of Aloe pectin. Polygalacturonic Aloe Pectin CitrusPectin Citrus Pectin acid Ca++ gel Yes Yes No Yes formation DM LM(<50%)LM(28%) HM(64%) 0

TABLE 15 Effect of endo-polygalacturonase on cold gelation of Aloepectin in the presence of monovalent cation (NaCI).Endo-polygalacturonase (unit/ml) 0 0.053 0.105 0.21 0.42 Gel formationFirm gel Firm gel Soft gel Softer gel Liquid

EXAMPLE 15 Use of Monovalent Cation-Based Aloe Pectin Gel as A NewMatrix for Antigen and Antibody Precipitation Reaction

The precipitation assay is a common diagnostic method for detectingpathogen-specific antigens or antibodies. It involves carefully layeringthe antigen solution over the antibody solution or vice versa. Thelayering step is important and care must be taken not to cause anydisturbance between the two solutions. The formation of a whiteprecipitation line between the two solutions as the result of diffusionindicates a positive result. Alternatively, this assay is performed inagar, which is referred as the agar diffusion assay. This assay involvespreparation of agar and takes a longer time to see results. The abilityof HMW Aloe pectin to form a gel in PBS at 4° C. provides an opportunityto design a new, simpler assay. One solution is kept in the solid stateat 4° C. so that another solution can be layered on top of it easily andconsistently. When the gel is brought back to room temperature, itchanges back to a non-viscous solution, allowing the diffusion to occur.

To test this potential usage, mouse IgG (antigen) and anti-mouse IgGantibodies (antibody) were used. 10 μl of the antigen at variousconcentrations was mixed with 0.4 ml of 1mg/ml AP 97-1 in PBS. The tubeswere then kept on ice and when a gel formed, 200 μl of the antibodysolution in PBS was directly added onto the gel. The tubes were thenreturned to room temperature. After 30 min, a precipitation lineappeared between the two solutions. When the antigen was added to theantibody solution with or without pectin at room temperature, no, oronly diffused, precipitation lines were observed. This suggests thatusing the Aloe pectin gel as a matrix will not only simplify suchantigen and antibody precipitation tests, but may also enhance theirsensitivity.

EXAMPLE 16 Extraction by Supercritical Fluid

The cell wall fibers (as obtained in Example 3) are packed into thesupercritical fluid (SF) extraction cell which is then sealed. The SFgenerator is turned on and upon reaching the desired conditions oftemperature and pressure, the SF is pumped into the extraction cell atthe appropriate flowrate. The pectin-rich exiting fluid is allowed tocool in the decompression chamber. The cooled fluid is then treated toisolate the pectin. One isolation method is to precipitate the pectin bythe addition of a water-soluble organic solvent, preferably ethanol, tothe fluid or partially evaporated fluid. The precipitated material isthen separated by filtration or centrifugation and dried. The pectin canalso be isolated from the fluid by removal of the fluid through freezedrying or evaporation. The fluid to be used for the SF extraction may bewater or an aqueous solution containing an acid or a base or a buffersalt or a water-soluble organic modifier or any combination of thepreceding additives. The process can be operated at temperatures betweenabout 300° C. and about 800° C. and at pressures between about 200 atm.and about 1000 atm.

EXAMPLE 17 Extraction of Aloe Pectins with Enzymes

Cell wall fibers are washed with water and suspended at a properconcentration in a buffer permitting the maximum activity of theenzyme(s) to be used. The enzymes that can be used includeendo-arabinase, endo-galactanase, and rhamnogalacturonase. Theendo-polygalacturonase, while usable for the naturally HM pectins, isnot suitable for Aloe pectin since it is a naturally LM one. The enzymeis then added. The fiber suspension is kept at 20-37° C. for certainperiod oftime (1-24 hrs). Remaining fibers are removed by filtration.Pectins are precipitated with alcohol and dried.

EXAMPLE 18 Extraction of Aloe Pectins with Microbes

Cell wall fibers are washed with water and suspended in water at aproper concentration. Microbes, either bacteria or fungi, that produceenzymes capable of liberating pectins from cell walls, are added to thefiber suspension. Bacillus subtilis is one example of such bacteria. Theenzymes produced include endo-arabinase, endo-galactanase,endo-polygalacturonase, and/or rhamnogalacturonase. The microbesproducing mainly endo-polygalacturonase is avoided since the Aloe pectinis naturally LM pectin. The extraction lasts for certain period of time(5-24 hrs) at 20-37° C. The remaining fibers were removed by coarsefiltration. The filtrate is then passed through a fine filter to removethe microbes. The final filtrate is mixed with alcohol (ethanol). Thepectin precipitates are collected and dried.

EXAMPLE 19 Use of Monovalent Cation-Based Gel as a Storage Matrix forPharmacological Agent

Pharmacological agents are often stored in buffered or non-bufferedphysiological saline (0.15 M NaCl) at 0-8° C. A pharmacological agent isany material that exerts a physiological effect on a biological system,either an animal or a plant. One problem often encountered by thisstorage form is aggregate formation and precipitation over time.

The monovalent cation-based thermo-reversible gel of Aloe pectin can beformed under physiological conditions (0.15 M NaCl, pH 7.4) at lowtemperature (4° C.) with a very low pectin concentration (1 mg/ml).Incorporation of pharmacological agents in the gel would provide amatrix which would reduce the opportunity for aggregation. Themonovalent cation-based gel quickly returns to solution once returned toRT (22° C.) so that the stored agent can be used in solution form.

The model protein bovine serum albumin (BSA) dissolved in physiologicalsaline (0.15 M NaCl) was mixed with an equal volume of 2 mg/ml of Aloepectin in 0.15 M NaCl. The final BSA concentration was 20 mg/ml and thefinal Aloe pectin concentration was 1 mg/ml. The mixture was then kepton ice. After 15 min, a gel formed. This indicated that the monovalentcation-based gel has the capacity for high concentrations of biologicalagents.

Black india ink is made of tiny carbon particles that tend toprecipitate over time when the suspension is left undisturbed. Todemonstrate the ability of the monovalent cation-based gel to preventprecipitation, black india ink was introduced into the gel system. Theblack india ink (Higgins, Faber-Castell Corporation, NJ) was diluted1000 times in 0.15 M NaC1. The diluted ink was the mixed with equalvolumes of 2 mg/ml Aloe pectin in 0.15 M NaC1 or with the 0.15 NaC1 onlyas a control. The mixtures were placed on ice. The mixture with pectinquickly formed a gel and the control remained a solution. Both sampleswere then stored at 4° C. After 48 hrs, it was evident that the upperportion of the control solution was less dark as compared to the lowerportion and a dense black area had formed at the bottom. This indicatedthat precipitation of the india ink particles had occurred. On the otherhand, the gel was evenly dark and no dense black area was observed atthe bottom of the tube. When the gel changed back to solution whenreturned to RT, the solution was also uniformly dark. This indicatedthat the gel could prevent the precipitation of the agents that mayresult from aggregation.

EXAMPLE 20 Physical and Chemical Characterization of Aloe Pectins

Appearance of the Final Products and Solutions. The dried Aloe pectinderived from pulp fibers, had an off white appearance. This color was insharp contrast to current commercial pectins of both citrus and appleincluding polygalacturonic acid prepared from citrus pectin, and otherpectins currently being developed such as sunflower pectin. Both appleand sunflower pectins are tan and citrus pectins have a lightyellow-brown color. The superior color quality of Aloe pectin from thepulp is likely due to the clear and color-free nature of the pulp.

When dissolved in water, the Aloe pectin solutions at a concentration of5 mg/ml were essentially clear, whereas the commercial ones were cloudyto various extents with the apple pectins being the cloudiest. Thisobservation was confirmed by measuring the absorbance at 600 nm (Table16). The absorbance at 600 mnm of Aloe pectins extracted from pulpfibers was at least 2 fold lower than any other pectins.

The Aloe pectins from green rind fibers exhibited a light green-brownpowder color to an extent similar to that of citrus pectins. Itssolution was less clear compared to the pulp pectins, but was as clearas the best citrus pectins (Table 16). TABLE 16 The cloudiness of pectinsolutions in water as measured at OD 600 nm. Source Pectins (5 mg/ml inwater) OD 600 nm Aloe pulp AP 10679 0.028 AP 97-1 0.044 Aloe rind APrind B1 (RT) 0.084 AP rind B1-2 (HT) 0.110 Commercial Citrus (LM), Sigma0.103 Citrus (HM), Sigma 0.082 Citrus, Sigma 0.176 Citrus, Spectrum0.136 Citrus, HF 0.272 Apple, HF 0.345 Polygalacuronic acid 0.206(citrus) Sigma

Features Overview

When compared to other pectins, Aloe pectin exhibited some uniquefeatures. First, Aloe pectin had a much higher rhamnose content. Thiswas shown with both unpurified and purified Aloe pectin in comparison tocommercial pectins and experimental pectins reported in the literature(Tables 10 and 11). This was also shown when the extraction conditionswere taken into consideration, i.e., Aloe pectin extracted with EDTA wascompared to other pectins extracted in a similar manner (with achelating agent) (Table 12). The rharnose content in Aloe pectin is morethan 3 fold higher in unpurified samples or more than 2 fold higher inpurified samples compared to the corresponding forms of other pectins.This difference was further substantiated by the fact that rhamnose/GalA ratios in Aloe pectin were similarly higher. Rhamnose, being abackbone sugar, has a critical effect on the backbone chain flexibility;the more rhamnose present, the more flexible the molecule will be. Thus,Aloe pectins are expected to be more flexible as compared to otherpectins. This may give Aloe pectin some distinct Theological properties.

Aloe pectins also contained a rare sugar, 3-OMe-rhamnose (Table 7). Itwas detected in all samples including the purified AP 10679. Itaccounted for ˜10% of the total rhamnose. The presence of this modifiedsugar has not been reported in any other pectins. The Aloe pectins ofthe present invention are relatively free of fiber. The fiber contentsof the Aloe pectins so obtained are less than about 20% by weight,preferably less than about 5% by weight, and even more preferably lessthan about 1% by weight.

A summary of other properties of Aloe pectins extracted from crude pulpand rind fibers are given in Table 17. TABLE 17 Overview of theproperties of Aloe pectins extracted from crude pulp and rind fibers.Gal A Intrinsic Content Fiber Extraction Powder Solution Viscosity (%,Phenol Ca++ Na+ Source Temp. Color Clearness MW (η, ml/g) w/w) DM DAc(%, w/w) Gelation Gelation AP B16 Pulp RT Off white Clear 1.06 × 10⁶ 55093 11 ND¹ <0.03 + + AP B16-2 Pulp HT Off white Clear  6.08 × 10⁵⁶ 337 9218 ND <0.03 + ±² AP 97-1 Pulp RT/HT Off white Clear 1.36 × 10⁶ 740 914.4 9.1 <0.03 + + AP rind B1 Rind RT Light brown³ Cloudy³ ND 978 81 4.0ND 0.045 + + AP rind B1-2 Rind HT Light brown Cloudy ND 523 84 9.5 ND0.041 + ± AP rind B2 Rind RT Light brown Cloudy ND 846 75 ND ND 0.219 ++¹Not determined.²Soft gel.³The color and cloudiness can be significantly reduced by additionalalcohol rinse.

While composition of isolated Aloe pectins and preferred methods forobtaining and using them have been disclosed, it will be apparent tothose skilled in the art that numerous modifications and variations arepossible in light of the above teaching. It should also be realized bythose skilled in the art that such modifications and variations do notdepart from the spirit and scope of the invention as set forth in theappended claims.

1. A method of using a purified Aloe pectin comprising mixing the pectinwith a pharmacological agent to form a pharmaceutical composition, andadministering the pharmaceutical composition to an animal, wherein thepurified Aloe pectin has a degree of methylation of 17.5% or less, andan average molecular weight of 6.08×10⁵ Daltons or higher.
 2. The methodof claim 1, wherein the animal is a human.
 3. The method of claim 1,wherein the purified Aloe pectin has an average molecular weight greaterthan about 1.0×10⁶ Daltons.
 4. The method of claim 1 wherein thepurified Aloe pectin has a degree of methylation of less than about 10%.5. A method of using a purified Aloe pectin comprising mixing the pectinwith a pharmacological agent to form a pharmaceutical composition, andadministering the pharmaceutical composition to an animal, wherein thepurified Aloe pectin has an average molecular weight of at least about6.08×10⁵ daltons.
 6. The method of claim 5, wherein the animal is ahuman.
 7. The method of claim 5, wherein the purified Aloe pectin has anaverage molecular weight greater than about 1.0×10⁶ Daltons.
 8. Themethod of claim 5, wherein the purified Aloe pectin has a degree ofmethylation of less than about 10%.
 9. A method of using a purified Aloepectin comprising mixing the pectin with a pharmacological agent to forma pharmaceutical composition, and administering the pharmaceuticalcomposition to an animal; wherein the pectin comprises 3-OMe Rhamnose atgreater than about
 0. 1% by mole and wherein the pectin is capable ofcalcium gel formation when present at a concentration of 0.2% (w/v) inan aqueous solution comprising 2 mM calcium chloride.
 10. The method ofclaim 9, wherein the animal is a human.
 11. The method of claim 9,wherein the purified Aloe pectin has an average molecular weight greaterthan about 1.0×10⁶ Daltons.
 12. The method of claim 11, wherein thepurified Aloe pectin has a degree of methylation of less than about 10%.13. A process for obtaining a purified Aloe pectin comprising: a)homogenizing an aloe vera plant or a portion thereof to give ahomogenized material; b) separating cell wall fibers from thehomogenized Aloe material; and c) extracting the pectin from theseparated cell wall fibers;
 14. The process of claim 13 wherein only aportion of the aloe vera plant is homogenized and the portion is thegreen rind of an aloe vera leaf.
 15. The process of claim 13 whereinonly a portion of the aloe vera plant is homogenized and the portion isthe inner gel of an aloe vera leaf.
 16. The process of claim 13 whereinthe separation of cell wall fibers comprises acidifying the homogenizedmaterial, and extracting with ethanol to produce a solid precipitatecomprising the cell wall fibers.
 17. The process of claim 16, whereinthe solid precipitate is treated with water, and the water insolubleportion comprising the cell wall fibers is separated from the water, andthe cell wall fiber are then extracted in step c.
 18. The process ofclaim 13 wherein the extraction employs a chelating agent.
 19. Theprocess of claim 18 wherein the chelating agent is EDTA.
 20. The processof claim 13 wherein the extraction occurs at a pH from about 7 to about9.
 21. The process of claim 13 wherein the extraction occurs at aboutroom temperature.
 22. The process of claim 13 wherein the purified Aloepectin has an average molecular weight greater than about 1.0×10⁶Daltons.
 23. The process of claim 22 wherein the purified Aloe pectinhas a degree of methylation of less than about 10%.
 24. A process forobtaining a purified aloe pectin comprising: a) homogenizing an aloevera plant or a portion thereof to give a homogenized material; b)separating cell wall fibers from the homogenized Aloe material; and c)extracting the pectin from the separated cell wall fibers; wherein thepurified aloe pectin has an average molecular weight of at least about6.08×10⁵ daltons.
 25. The process of claim 24 wherein only akportion ofthe aloe vera plant is homogenized and the portion is the green rind ofan aloe vera leaf.
 26. The process of claim 24 wherein only a portion ofthe aloe vera plant is homogenized and the portion is the inner gel ofan aloe vera leaf.
 27. The process of claim 24 wherein the separation ofcell wall fibers comprises acidifying the homogenized material, andextracting with ethanol to produce a solid precipitate comprising thecell wall fibers.
 28. The process of claim 27, wherein the solidprecipitate is treated with water, and the water insoluble portioncomprising the cell wall fibers is separated from the water, and thecell wall fibers are then extracted in step c).
 29. The process of claim28 wherein the extraction employs a chelating agent.
 30. The process ofclaim 29 wherein the chelating agent is EDTA.
 31. The process of claim28 wherein the extraction occurs at a pH from about 7 to about
 9. 32.The process of claim 28 wherein the extraction occurs at about roomtemperature.
 33. The process of claim 24 wherein the purified Aloepectin has an average molecular weight greater than about 1.0×10⁶Daltons.
 34. The process of claim 33 wherein the purified Aloe pectinhas a degree of methylation of less than about 10%.
 35. A process forobtaining a purified aloe pectin comprising: a) homogenizing an aloevera plant or a portion thereof to give a homogenized material; b)separating cell wall fibers from the homogenized Aloe material; and c)extracting the pectin from the separated cell wall fibers; wherein thepectin comprises 3-OMe Rhamnose at greater than about 0.1% by mole andwherein the pectin is capable of calcium gel formation when present at aconcentration of 0.2% (w/v) in an aqueous solution comprising 2 mMcalcium chloride.
 36. The process of claim 35 wherein only a portion ofthe aloe vera plant is homogenized and the portion is the green rind ofan aloe vera leaf.
 37. The process of claim 35 wherein only a portion ofthe aloe vera plant is homogenized and the portion is the inner gel ofan aloe vera leaf.
 38. The process of claim 35 wherein the separation ofcell wall fibers comprises acidifying the homogenized material, andextracting with ethanol to produce a solid precipitate comprising thecell wall fibers.
 39. The process of claim 38, wherein the solidprecipitate is treated with water, and the water insoluble portioncomprising the cell wall fibers is separated from the water, and thecell wall fiber are then extracted in step c).
 40. The process of claim35 wherein the extraction employs a chelating agent.
 41. The process ofclaim 40 wherein the chelating agent is EDTA.
 42. The process of claim39 wherein the extraction occurs at a pH from about 7 to about
 9. 43.The process of claim 35 wherein the extraction occurs at about roomtemperature.
 44. The process of claim 35 wherein the purified Aloepectin has an average molecular weight greater than about 1.0×10⁶Daltons.
 45. The process of claim 35 wherein the purified Aloe pectinhas a degree of methylation of less than about 10%.